Protein folding is a problem of the utmost basic and medical relevance, with important implications on our understanding of protein structure, function, evolution and regulation. A better understanding of the cellular pathways of protein folding will have important therapeutic applications impacting many devastating human diseases. The transformation of the one-dimensional genetic information into three-dimensional protein structures depends on the accuracy and efficiency of the process of protein folding. Folding in the cell differs in three fundamental ways from the in vitro refolding experiments used to understand the principles of chaperone mechanism and action under reductionistic paradigms. Firstly, in the cell protein folding must be accomplished in the context of the vectorial synthesis of polypeptide chains on ribosomes. In principle, the N-terminal portion of the nascent polypeptide could fold spontaneously as it emerges from the ribosome. Secondly, it must take place in a crowded milieu, which heightens the chances of aggregation for unfolded polypeptides. Given the high density of folding nascent chains emerging from polysomes and since unfolded or partially folded polypeptide chains have a strong tendency to aggregate, it is critically important that the growing polypeptide is effectively prevented from misfolding and aggregating until a chain length suitable for productive folding has been synthesized. Thirdly, the cell contains many different chaperones, as well the ubiquitin-proteasome degradation pathway, all of which are presumably vying for access to non-native protein species. In the previous funding period we established that eukaryotic cells possess a complex chaperone machinery that associates with translating ribosomes, and appears dedicated to protein biogenesis. We aim to understand the principles and mechanisms by which these molecular chaperones interact with, and stabilize, nascent polypeptides and assist their folding in vivo. The major thrusts of the grant have been, and continue to be: (i) to integrate in vivo and in vitro approaches to define the substrates and function of cotranslationally acting chaperones; (ii) to gain a mechanistic understanding of how these chaperones bind to and fold nascent polypeptides and (iii) to understand the basis of chaperone interactions with the translational machinery. In addition, we are beginning to explore the role of mRNA sequence (both codon choice and UTR regions) in determining the fate of the nascent polypeptide. RELEVANCE (See instructions): The transformation of the one-dimensional genetic information into three-dimensional protein structures depends on the accuracy and efficiency of the process of protein folding. Failure of correct protein folding is associated with an increasing list of human maladies, ranging from neurodegeneration to cancer. The long term goal of this program is to understand the process of protein folding as it occurs in the cell, where proteins emerge vectorially upon translation in the ribosome. We examine the underlying logic and mechanisms of cotranslational protein folding and the function of molecular chaperones and quality control components in this process.